WO2009145333A1 - Method and unit for detecting interaction of biological molecules - Google Patents

Abstract

Disclosed is a unit for detecting the interaction of biological molecules using a carrier whereon the biological molecules are affixed. The unit has a hollow holder that has an open part at one end, the other end of which is closed, and a carrier support member to which is affixed a carrier whereon biological molecules are affixed that can be inserted into the hollow holder. By the rear end part of the carrier support member and the edge of the open part of the hollow holder engaging when the carrier support member is inserted into the hollow holder, the hollow holder is sealed, and the carrier support member and the hollow holder are positioned. In a cross‑sectional view cut along a plane that includes the axial center of the hollow holder when positioned, the surface area delineated by the inside of the hollow holder and the outside of the carrier support member to which the carrier is affixed is approximately the same on the left and right of the axial center, in the region from where the carrier is affixed to the tip part of the carrier support member.

Description

 Method and unit for detecting interactions of biologically relevant molecules

 The present invention relates to a method for detecting an interaction of biologically relevant molecules using a carrier on which biologically relevant molecules are immobilized, and a unit for use in the method.

 Light

 Background art

 Advances in genome analysis have elucidated biological molecules involved in the physiological book reaction of various organisms. These bio-related molecules include DNA, proteins, sugar chains, cells, etc., whose functions and structures have been elucidated are used in various industrial applications such as drug discovery, clinical testing, food testing, and environmental testing. The

 The following methods are commonly used in clinical tests and other tests. In other words, when a sample is brought into contact with a device in which a probe molecule (hereinafter referred to as a ligand) that specifically binds to a biologically relevant molecule (hereinafter referred to as an analyte) to be detected is immobilized on a carrier, In the presence of the analyte, the captured analyte is detected as it binds to the ligand and is captured on the support.

 Even in the inspection methods as described above, in recent years, high speed and automation have been demanded, and a detection method that comprehensively measures several hundred to several tens of thousands of biologically relevant molecules has been demanded. Device integration using the so-called MEMS technology, so-called MEMS technology, is possible, and it is used as a so-called microarray for comprehensive analysis in drug discovery research and biotechnology research.

 Depending on the type of probe molecules immobilized on the carrier, microarrays as devices include DNA microarrays (also called DNA chips), protein microarrays (also called protein chips), cell microarrays (also called cell chips), etc. There is.

For analysis, for example, an analyte contained in the specimen is detected by contacting the specimen that has been fluorescently labeled with a fluorescent substance in advance, and then detecting and measuring the fluorescent signal emitted by the fluorescent substance after washing the microarray. Identify or quantify (Special Table 2) 0 0 6-5 1 5 0 6 5).

 A microarray such as a DNA chip is usually a slide glass-like size, and the sample is suspended on it and covered with a slide. In the future, it will be necessary to automatically process a large number of microarrays depending on the application. In that case, miniaturization of the microarray is desired, and further development of an efficient means for automatically processing the miniaturized microarray is desired. Summary of the Invention

 In order to automatically process a miniaturized microarray, the present inventors fixed a microarray on a support member, inserted it into a hollow holder containing a sample, and reacted it. Since the volume was very small (several to several hundred μ), it was found that the contact between the specimen and the microarray was not sufficiently performed due to the effect of surface tension, resulting in insufficient reaction. Accordingly, an object of the present invention is to provide a means for sufficiently contacting a small amount of specimen with a microarray in automatic processing of a miniaturized microarray.

 The present inventors fixed the microarray on a support member, and when inserting it into a hollow holder containing a reaction solution and reacting it, positioning is performed so that the support member is inserted into the hollow holder without deviation. As a result, it was found that the specimen and the microarray were sufficiently brought into contact with each other, and the present invention was completed.

 That is, the present invention includes the following inventions.

 (1) A hollow holder for detecting the interaction of biological molecules using a carrier on which biological molecules are immobilized, the hollow holder having an opening at one end and the other end closed. And a carrier support member in which a carrier on which a bio-related molecule is immobilized is fixed and can be inserted into the hollow holder, and the carrier support member is inserted into the hollow holder. By engaging the rear end of the carrier support member with the edge of the hollow holder opening, the hollow holder is sealed and the carrier support member and the hollow holder are positioned. In the sectional view taken along the plane including the shaft center, the area force defined by the inside of the hollow holder and the outside of the carrier support member on which the carrier is fixed, the area from the carrier fixing part to the tip part of the carrier support member And on the left and right sides of the axis Identical, pre-unit.

(2) The carrier fixing portion in the carrier support member is a recess having a bottom surface and a side surface, The unit according to (1), wherein is disposed on a bottom surface of the recess.

 (3) The unit according to (2), wherein at least a side surface on the rear end side of the concave portion of the carrier support member is an inclined surface.

 (4) The unit according to (3), wherein the surface roughness of the inclined surface is 10 / X m or less, and the angle formed between the inclined surface and the bottom surface is 75 ° or less.

 (5) The unit according to any one of (2) to (4), wherein a waste liquid groove is formed from the side surface on the tip end side of the carrier support member recess to the tip end portion.

 (6) When the carrier support member is inserted into the hollow holder, the ratio of the volume of the carrier support member to the volume of the hollow holder is 60% or more in the region closer to the tip than the carrier fixing portion of the carrier support member. The unit according to any one of (1) to (5).

 (7) In the posture in which the carrier support member is inserted into the hollow holder, the ratio of the volume of the carrier support member to the volume of the hollow holder is 25 to 70%. (1) to (6) The unit.

 (8) Each has a plurality of carrier support members to which the carrier is fixed, each rear end portion of the carrier support member is fixed to a face material, and each has a plurality of hollow holders corresponding to each support member. The unit according to any one of (1) to (7).

 (9) A method for detecting the interaction of biological molecules using a carrier on which biological molecules are immobilized, which has an opening at one end and is closed at the other end to contain a reaction solution. By inserting a carrier support member on which a carrier on which biological molecules are immobilized is inserted into a hollow holder, the biological molecules on the carrier and the fluorescently labeled biological molecules in the reaction solution are combined. Interaction process for interaction, washing process for removing the carrier-related molecules that did not interact with the carrier-related molecules immobilized on the carrier by irradiating the carrier with excitation light, Including a detection step of detecting fluorescence, and in a posture in which the carrier support member is inserted into the hollow holder, the rear end portion of the carrier support member and the edge of the hollow holder opening are engaged, whereby the hollow holder is hermetically sealed. With The carrier supporting member and the hollow holder are positioned in the cross-sectional view taken along the plane including the axial center of the hollow holder in the positioning posture, and the side of the hollow holder and the outer side of the carrier supporting member on which the carrier is fixed The method, wherein the defined areas are substantially the same on the left and right sides of the axis in the region from the carrier fixing part to the tip part of the carrier support member.

(1 0) The carrier fixing portion in the carrier supporting member is a recess having a bottom surface and a side surface, The method according to (9), wherein the body is disposed on the bottom surface of the recess.

 (11) The method according to (10), wherein at least the side surface on the rear end side of the carrier supporting member recess is an inclined surface.

 (1 2) The method according to (1 1), wherein the surface roughness of the inclined surface is 10 / z m or less, and the angle formed by the inclined surface and the bottom surface is 75 ° or less.

 (13) The method according to any one of (10) to (12), wherein a waste liquid groove is formed from the side surface on the distal end side of the concave portion of the carrier support member to the distal end portion.

 (14) In the posture in which the carrier support member is inserted into the hollow holder, the ratio of the volume of the carrier support member to the volume of the hollow holder in the region on the tip side of the carrier fixing portion of the carrier support member is 60. % Or more, The method in any one of (9)-(1 3).

 (15) In the posture in which the carrier support member is inserted into the hollow holder, the volume ratio of the carrier support member to the volume of the hollow holder is 25 to 70%. (9) to (14) Either method.

 (16) Each of the carrier support members has a plurality of carrier support members to which the carrier is fixed, the rear end portions of the carrier support members are each fixed to a face material, and a plurality of hollow holders corresponding to the respective support members. The method according to any one of (9) to (15).

 The present invention provides a means for sufficiently contacting a small amount of specimen with a microarray in automatic processing of a miniaturized microarray.

 This specification includes the contents described in the description, claims and drawings of Japanese Patent Application No. 20 08-1 4 1 1 5 5 which is the basis of the priority of the present application. Brief Description of Drawings

 FIG. 1 shows an embodiment of the present invention.

 FIG. 2 shows an embodiment of the present invention.

 FIG. 3 shows an embodiment of the present invention.

 FIG. 4 shows an embodiment of the present invention.

 FIG. 5 shows an embodiment of the present invention.

 FIG. 6 shows a result of comparison between the case where the rear end portion of the carrier support member is engaged with the edge of the hollow holder opening (a) and the case where it is not engaged (b).

FIG. 7 shows a carrier support member (a) having a waste liquid groove and a carrier support member having no waste liquid groove. One embodiment of (b) and hollow holder (c) is shown.

 FIG. 8 shows the detection results when the carrier support member having the waste liquid groove, the tray without the waste liquid groove, and the carrier support member are used.

 Fig. 9 shows a carrier support member (a) in which the proportion of the volume of the carrier support member occupying the volume of the hollow holder is 80% and a carrier support member (b) in which the volume is 50%. The results are shown in which each was inserted into a hollow holder containing the reaction solution. BEST MODE FOR CARRYING OUT THE INVENTION

 In the present invention, biologically relevant molecules include nucleic acids such as DNA and RNA, peptides, sugar chains and cells, complexes thereof, and complexes of these with other molecules. In the present invention, peptides include oligopeptides, polypeptides, and proteins. When the biologically relevant molecule immobilized on the carrier is a peptide, a peptide of usually 1 to 1000 kDa, preferably 1 to 200 kDa is suitable. When the biologically relevant molecule immobilized on the carrier is a nucleic acid, a nucleic acid usually having 3 to 5000 bases, preferably 10 to 1000 bases, is suitable. When the biologically relevant molecule immobilized on the carrier is a sugar chain, a sugar chain of 1 to 100 sugars, preferably 1 to 30 sugars is preferable. In the present invention, the biologically relevant molecule is preferably a nucleic acid, more preferably DNA.

 The interaction of biologically relevant molecules preferably refers to a specific interaction between biologically relevant molecules, for example, interactions between proteins, interactions between proteins and peptides, interactions between nucleic acids, interactions between proteins and nucleic acids. Effects, and interactions between proteins and compounds. More specifically, hybridization between nucleic acid complementary strands, reaction between antigen and antibody or fragment thereof, binding reaction between enzyme and substrate or inhibitor, binding reaction between ligand and receptor, binding between avidin and piotin Reaction, Binding reaction between nucleic acid and transcription factor, Binding reaction between cell adhesion factor, Binding reaction between sugar chain and protein, Binding reaction between fatty chain and protein, Binding reaction between phosphate group and protein, Binding reaction between prosthetic factor and protein And so on.

The present invention relates to a detection of an interaction between biological molecules using a carrier (also referred to as a microarray) on which biological molecules are immobilized, and a hollow having an opening at one end and a closed end. The holder and the microarray are fixed. A unit having an insertable carrier support member is used. By inserting the carrier support member into a hollow holder containing the reaction solution, the microarray and the reaction solution are brought into contact with each other, and the biologically relevant molecules are brought into contact with each other. Interact.

 The hollow holder having an opening at one end and closed at the other end is not particularly limited as long as it can accommodate the reaction solution. The hollow holder may be a single holder or a plurality of hollow holders connected together. Examples of the hollow holder include a microphone mouth tube and a microtiter plate (for example, 96 well plate) that are usually used in this technical field. The dimension of the hollow holder can be appropriately set by those skilled in the art depending on the application. For example, the tube holder has a diameter in the range of 6 to 12 mm and a length of 15 to 35 mm. Can be used.

 The hollow holder contains a specimen containing a fluorescently labeled biological molecule as a reaction solution, and a carrier support member is inserted into the hollow holder so that the carrier and the reaction solution are brought into contact with each other. And fluorescently labeled biologically relevant molecules in the reaction solution are allowed to interact. A nucleic acid amplification product may be accommodated as a reaction solution. In the washing process of removing the biological molecules that did not interact with the biological molecules immobilized on the carrier by washing the carrier, the washing liquid is accommodated in the hollow holder, and the carrier supporting member is used as the carrier support member after the interaction. It can also be cleaned by inserting it. A hollow holder containing a specimen as a reaction solution and a hollow holder containing a cleaning solution as a reaction solution may be used.

In the interaction step in which the biologically relevant molecule on the carrier interacts with the fluorescently labeled biologically relevant molecule in the reaction solution, it is preferable to heat the reaction solution by heating the hollow holder. In the present invention, in the posture in which the carrier support member is inserted into the hollow holder, the hollow holder is sealed by the engagement of the rear end portion of the carrier support member and the edge of the hollow holder opening. In addition, the evaporation of the reaction solution can be suppressed and heating can be performed efficiently. In some cases, the reaction liquid partially evaporated upon heating is cooled at the rear end of the carrier support member that closes the hollow holder to form droplets. Therefore, in the interaction process, heating the rear end of the carrier support member together also suppresses the formation of droplets, reduces the amount of reaction liquid, and changes the concentration of biological molecules in the reaction liquid. Can be prevented. The heating temperature in the interaction step is 30 to 60 ° C., preferably 35 to 55 ° C., and the heating temperature at the rear end of the carrier support member is the same. In the present invention, in a posture in which the carrier support member is inserted into the hollow holder, the carrier support member and the hollow holder are positioned by engaging the rear end portion of the carrier support member with the edge of the hollow holder opening. Is done. For example, as shown in FIG. 1, the carrier supporting member 12 is engaged with the rear end portion 13 of the support member 1 2 and the edge 15 of the opening of the hollow holder 11 1 so that the support member is placed inside the hollow holder. (Fig. La), but if the rear end portion 13 of the carrier support member 12 and the edge 15 of the opening of the hollow holder 11 are not engaged, the carrier support member The position of the carrier support member in the hollow holder changes as the holder is inserted into the hollow holder, and cannot be positioned at a predetermined position (FIG. 16).

 In the present invention, in the above-described positioning posture in which the carrier support member is inserted into the hollow holder, the carrier support in which the inside of the hollow holder and the carrier are fixed in a cross-sectional view taken along the plane including the axis of the hollow holder. The carrier support member and the hollow holder are configured so that the area defined by the outside of the member is substantially the same on the left and right sides of the shaft center in the region from the carrier fixing part to the tip part of the carrier support member. The

For example, FIG. 2 shows an example of a cross-sectional view taken along a plane including the axis 21 of the hollow holder in the above-mentioned positioning posture in which the carrier support member is inserted into the hollow holder. In this sectional view, of the area defined by the inside of the hollow holder 1 and the outside of the carrier support member on which the carrier is fixed, the area of the region from the carrier fixing part to the tip 24 of the carrier support member is The left side of the axis is the area of the part represented by 2 2, and the right side of the axis is the area of the part represented by 2 3. By configuring the carrier support member and the hollow holder so that the area of 22 and the area of 23 are substantially the same, the left and right surface tensions become equal, and the liquid level of the reaction liquid is not biased. The carrier 14 can be sufficiently brought into contact with the reaction solution. In Fig. 2a, the area of 2 2 and the area of 2 3 are not the same, so the reaction solution is biased as shown by the dotted line. On the other hand, in FIG. 2b, the area of 2 2 and the area of 2 3 are almost the same, so the reaction solution becomes aqueous. “Substantially the same” means that the difference is 20% or less, preferably 10% or less, more preferably 5% or less. Fig. 2 shows a cross-sectional view in which the carrier 14 is cut out of a cross-sectional view taken along the plane including the axis 21 of the hollow holder, and is therefore not symmetrical with respect to the axis of the hollow holder. However, the same applies to the cross section that is symmetrical with respect to the axis of the hollow holder. That is, the left and right areas are substantially the same in any cross section including the axis of the hollow holder. In a preferred embodiment, for example, as shown in FIG. 3, the carrier fixing portion 3 1 in the carrier supporting member is a recess having a bottom surface 3 2 and side surfaces 3 3 (3 3 a and 3 3 b), and the carrier 1 4 Is disposed on the bottom surface 32 of the recess. More preferably, at least the side surface 3 3 a on the rear end portion 13 side of the carrier supporting member recess 3 1 is an inclined surface. It is preferable that the side surface 3 3 b of the carrier support member recess 3 1 on the front end portion 2 4 side is also an inclined surface. By making the side surface 33a on the rear end side into an inclined surface, bubbles that can be mixed when the carrier support member is inserted into the reaction solution in the hollow holder can easily escape upward. The surface roughness of this inclined surface is 10 / zm or less, preferably 1 μιη or less, and the angle between the inclined surface and the bottom surface (34 in FIG. 3) is 75 ° or less, preferably 45 °. By making the following, bubbles can escape more easily. If the bubbles do not escape well, the contact between the carrier and the reaction solution may be hindered, and the interaction of organism-related molecules may be hindered by a part of the carrier, but by adopting the above configuration that allows the bubbles to escape well, Good contact between the support and the reaction solution can be achieved.

It is preferable that a waste liquid groove is formed on the carrier support member from the side surface on the tip end side of the recess to the tip end portion. For example, as shown in FIG. 4, the waste liquid groove 41 is formed from the side surface 33 b on the front end side of the recess to the front end 24, thereby lifting the carrier support member from the reaction liquid in the hollow holder. The reaction solution that can remain in the recesses can be efficiently drained. After pulling up the carrier support member, the tip portion 24 of the carrier support member is brought into contact with the filter paper, so that the waste liquid can be more efficiently drained. In the present invention, after the interaction of the bio-related molecule, in the cleaning step of removing the bio-related molecule that did not interact with the bio-related molecule immobilized on the carrier, a cleaning liquid containing a deliquescent substance is used. The carrier can be irradiated with excitation light as it is without drying, and fluorescence can be detected by a detector. Since a solution containing salt is used in the interaction and washing of biological molecules, when the carrier is dried, drying unevenness due to salt occurs, and scattered light due to drying unevenness is strong, making accurate detection difficult. There is a case. In particular, in the detector of the imaging optical system, scattered light due to drying unevenness is strong, and accurate detection may be difficult compared to a scanning detector, but a cleaning liquid containing a deliquescent substance is used. If the detection is performed without drying, the generation of scattered light due to uneven drying can be suppressed. Even in the case of detecting without drying the carrier, a detection error may occur if a liquid pool is formed in the concave portion of the carrier supporting member. However, by providing a waste liquid groove, excess cleaning liquid left in the concave portion is provided. As a result, the waste liquid can be drained immediately and detection errors can be reduced. The deliquescent material is not particularly limited as long as it does not inhibit the interaction of biological molecules. For example, alkaline earth metal salts such as magnesium chloride, calcium chloride and magnesium hydroxide, potassium carbonate, bromide Examples thereof include alkali metal salts such as sodium. The concentration of the deliquescent substance in the cleaning solution is usually from 0.01 to 3. O mo 1 Z l, preferably from 0.05 to 1: O mol Z l and more preferably from 0.2 to 0.5 mo 1 / 1.

 In a preferred aspect of the present invention, in the posture in which the carrier support member is inserted into the hollow holder, the carrier support member occupies the volume of the hollow holder in the region on the tip 24 side from the carrier fixing portion of the carrier support member. The volume ratio is 60% or more, preferably 80% or more, more preferably 90 to 95%. For example, in FIG. 5, in the posture in which the carrier support member is inserted into the hollow holder, the region on the tip side from the carrier fixing portion of the carrier support member 12 (the region below the dotted line), that is, the carrier fixing portion When the front end of the hollow holder is cut by a plane perpendicular to the axis of the hollow holder 11 1, the ratio of the volume of the carrier support member to the volume of the hollow holder on the side not including the carrier fixing portion is the above value. . By designing the tip side of the carrier support member to be larger than the carrier fixing part, when the carrier support member is inserted into the hollow holder, even if the reaction liquid contained in the hollow holder is very small, the liquid level As a result, it becomes possible to make sufficient contact with the carrier. Therefore, the amount of liquid during operation can be reduced.

 Further, in the posture in which the carrier support member is inserted into the hollow holder, the ratio of the volume of the carrier support member to the volume of the hollow holder is preferably 25 to 70%, more preferably 40 to 50%. . By setting the volume ratio of the carrier support member to a certain value or less, it is possible to prevent liquid from spilling when the carrier support member is inserted.

The unit used in the present invention may have a plurality of carrier support members and a plurality of hollow holders corresponding to the respective support members. The carrier is fixed to each of the plurality of carrier support members, and preferably the rear end portions of the carrier support members are fixed to the face material, respectively, so that they can operate as a unit. Having a plurality of hollow holders corresponding to each carrier support member means that the carrier support members are respectively inserted into the plurality of hollow holders. By using a plurality of carrier support members each having a carrier immobilized thereon and a plurality of hollow holders, a large amount of tests can be efficiently performed. In the detection method of the present invention, the carrier is washed to remove biologically related molecules that have not interacted with the biologically related molecules immobilized on the carrier, and then the carrier is irradiated with excitation light for detection. The detection process which detects fluorescence with a container is implemented.

 In the present invention, it is preferable to use a detector of an imaging optical system as the detector. The imaging optical system detector detects the intensity of the fluorescence obtained by irradiating the carrier with excitation light. The detector of the imaging optical system is usually a laser for irradiating excitation light, a fluorescence filter that transmits only the fluorescence of the target wavelength, and a light detector for detecting the fluorescence that has passed through the fluorescence filter ( For example, it has a CCD camera. In the present invention, usually, excitation light is obliquely irradiated on the entire carrier at once with a single laser, and fluorescence is detected from the front of the carrier. To irradiate the excitation light obliquely with respect to the carrier means that the angle formed between the carrier surface and the laser beam is less than 90 °, and is usually 30 to 70 °, preferably 40 to 6 Irradiate at an angle of 0 °. In the detector of the imaging optical system, it is not necessary to scan the entire surface of the carrier with a laser, and detection can be performed in a short time. Since the entire carrier is irradiated with excitation light at once, the carrier for immobilizing biological molecules is a relatively small carrier, for example, a size of 10 mm or less, preferably 5 mm or less, more preferably 3 Use a carrier of mm or less, most preferably 1 to 5 mm square.

As the material for the carrier for immobilizing the biologically relevant molecule, those known in the art can be used and are not particularly limited. For example, platinum, platinum black, gold, palladium, rhodium, silver, mercury, noble metals such as tungsten and their compounds, and conductor materials such as carbon, such as graphite, carbon fiber; single crystal silicon, amorphous Silicon materials such as silicon, silicon carbide, oxide silicon, and nitride nitride, and composite materials of these silicon materials such as SOI (silicon on insulator); glass, quartz glass, alumina, Inorganic materials such as sapphire, ceramics, forsterite, and photosensitive glass; polyethylene, ethylene, polypropylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride Polyacetic acid L, Polyvinyl alcohol, Poly buracetal, Acrylic resin, Polyacrylo-trinole, Polystyrene, Acetal resin, Polycarbonate, Polyamide, Phenolic resin, Urea resin, Epoxy resin, Melamine resin, Styrene Polymer, acrylonitrile 'butadiene styrene copolymer, polyphenylene Examples include organic materials such as lenoxide and polysulfone.

 In the present invention, a carrier having a force layer and a chemically modifying group on the surface is preferably used as the carrier. Carriers having a carbon layer and a chemical modification group on the surface include those having a carbon layer and a chemical modification group on the surface of the substrate, and those having a chemical modification group on the surface of the substrate made of the carbon layer. . As the material for the substrate, those known in the art can be used, and there is no particular limitation, and the same materials as those described above can be used. The present invention is suitably used for a carrier having a fine flat plate structure. Since it is easy to produce a carrier having a fine flat plate structure, it is preferable to use a substrate made of a silicon material or a resin material. In particular, a carrier having a carbon layer and a chemical modifying group on the surface of a substrate made of single crystal silicon. More preferred. Single-crystal silicon includes crystal parts that have slightly changed the orientation of the crystal axis (sometimes called mosaic crystals), and atomic scale disturbances (lattice defects). What is included is also included.

 The carbon layer to be formed on the substrate is not particularly limited, but synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond-like force), amorphous force, carbon-based material (for example, (Graphite, fullerene, carbon nanotube), a mixture thereof, or a laminate of them is preferably used. In addition, carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tantalum carbide, zirconium carbide, molybdenum carbide, chromium carbide, and vanadium carbide may be used. Here, soft diamond is a generic term for incomplete diamond structures that are a mixture of diamond and carbon, such as so-called diamond-like carbon (DLC), and its mixing ratio. Is not particularly limited. The carbon layer is excellent in chemical stability and can withstand subsequent reactions in the introduction of chemical modification groups and binding to biologically relevant molecules, and the binding is flexible due to electrostatic binding to biologically relevant molecules. It is advantageous in that it has sex. It is also advantageous in that nonspecific adsorption is small in the binding reaction with biologically relevant molecules. As described above, the substrate itself may be a carrier having a carbon layer force.

In the present invention, the carbon layer can be formed by a known method. For example, the microphone mouth plasma C VD (Ch ical vapordeposit) method, ECR and VD (E lectriccyclotronreson ancech em icalvapordeposit) method, ICP (I nductivecoupledpla sma) method, a DC sputtering method, ECR (E lectriccyclotronreson ance) scan Nono ⁰ Ttaringu method, Ion spoon evaporation, arc evaporation, laser deposition, Examples include EB (Electronbe am) vapor deposition and resistance heating vapor deposition.

 In the high-frequency plasma CVD method, the raw gas (methane) is decomposed by glow discharge generated between electrodes due to high frequency, and a DLC (diamond-like carbon) layer is synthesized on the substrate. In the ionization deposition method, the source gas (benzene) is decomposed and ionized using the thermoelectrons generated by the tungsten filament, and a bias layer is formed on the substrate by a bias voltage. The DLC layer may be formed by ionized vapor deposition in a mixed gas composed of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of the remaining methane gas. In the arc deposition method, a DC voltage is applied between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode) to cause an arc discharge in a vacuum and generate a plasma of carbon atoms from the cathode. Then, by applying a negative bias voltage to the substrate even more than the evaporation source, the carbon ions in the plasma can be accelerated toward the substrate to form a carbon layer.

 In laser vapor deposition, for example, a carbon layer can be formed by depositing carbon atoms on a glass substrate by irradiating and melting Nd: YAG laser (pulse oscillation) light on the target plate of the graph. .

 When the carbon layer is formed on the surface of the substrate, the thickness of the carbon layer is usually about a monomolecular layer to about 100 μιη. If it is too thin, the surface of the base substrate may be locally exposed. When the thickness is too thick, the productivity deteriorates, so the thickness is preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm.

 By introducing a chemical modifying group into the surface of the substrate on which the carbon layer is formed, the bio-related molecule can be firmly immobilized on the carrier. The chemical modification group to be introduced can be appropriately selected by those skilled in the art and is not particularly limited. For example, an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group, a metal chelate, and an active ester group may be used. Can be mentioned.

For example, the amino group is introduced by irradiating the carbon layer with ultraviolet rays in ammonia gas. Or by plasma treatment. Alternatively, the carbon layer can be chlorinated by irradiating with ultraviolet rays in chlorine gas and further irradiating with ultraviolet rays in ammonia gas. Alternatively, it can also be carried out by reacting with a chlorinated force layer in a polyvalent amine gas such as methylenediamine or ethylenediamine. The introduction of the carboxyl group can be carried out, for example, by reacting an appropriate compound with the carbon layer aminated as described above. Examples of the compound used for introducing a carboxyl group include, for example, the formula: X—R ¹ —COOH (wherein X is a halogen atom, R ¹ is a divalent hydrocarbon group having 10 to 12 carbon atoms) ) Halocarbonic acid such as chloroacetic acid, fluoroacetic acid, bromoacetic acid, sodoacetic acid, 2-chloropropionic acid, 3-propoxypropionic acid, 3_cloacrylic acid, 4-chloroform Benzoic acid; dicarboxylic acid represented by the formula: HOOC—R ² _COOH (wherein R ² represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), for example, oxalic acid, malonic acid , Succinic acid, maleic acid, fumaric acid, phthalic acid; polyacrylic acid, polymethacrylic acid, trimellitic acid, butanetetracarboxylic acid and other polyvalent carboxylic acids; formula: R ³ _CO -R ⁴ -COOH (wherein , R ³ is a hydrogen atom or a divalent hydrocarbon with carbon number 1-1 2 , R ⁴ is keto acid or aldehyde arsenate de acid represented by representing) a divalent hydrocarbon group of from 1 to 1 2 carbon atoms; ^{Formula:. X- OC- R 5 - COOH} ( wherein, X is a halogen atom , R ⁵ represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms.) Monocarboxylic acid monohalides such as succinic acid monochloride, malonic acid monochloride; phthalic anhydride, anhydrous And acid anhydrides such as succinic acid, oxalic anhydride, maleic anhydride, and butanetetracarboxylic anhydride.

 Introduction of an epoxy group can be carried out, for example, by reacting a suitable polyvalent epoxy compound with the carbon layer aminated as described above. Alternatively, it can be obtained by reacting an organic peracid with the carbon = carbon double bond contained in the carbon layer. Examples of organic peracids include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, and trifluoroperacetic acid.

 The introduction of the formyl group can be carried out, for example, by reacting glutaraldehyde with the carbon layer aminated as described above.

Introduction of a hydroxyl group can be carried out, for example, by reacting water with the carbon layer chlorinated as described above. The active ester group means an ester group having an electron-attracting group with high acidity on the alcohol side of the ester group and activating a nucleophilic reaction, that is, an ester group having a high reaction activity. An ester group having an electron-attracting group on the alcohol side of the ester group and activated more than an alkyl ester. The active ester group has reactivity with groups such as amino group, thiol group, and hydroxyl group. More specifically, phenol esters, thiphenol esters, N-hydroxyamine esters, cyanomethyl esters, and esters of heterocyclic hydroxy compounds are much higher than alkyl esters. It is known as an active ester group having activity. More specifically, the active ester group includes, for example, p-nitrophenyl group, N-hydroxysuccinimid group, succinic acid imide group, phthalic acid imide group, 5 _norbornene _ 2,3-dicad Examples include a lupoximid group, and an N-hydroxysuccinimid group is particularly preferred.

 The introduction of the active ester group is, for example, by converting the carboxyl group introduced as described above to cyanamide carpositimide (for example, 1_ [3- (dimethylamino) propyl] -3_ethylcarbodiimide). This can be carried out by active esterification with a compound such as a dehydration condensing agent and N-hydroxysuccinimide. By this treatment, a group in which an active ester group such as an N-hydroxysuccinimide group is bonded to the end of the hydrocarbon group via an amide bond can be formed (Japanese Patent Laid-Open No. 20 0 1- 1 3 9 5 3 2).

 When nucleic acids such as DNA and RNA are immobilized, it is preferable to introduce an amino group, an epoxy group, a carbodiimide group, a formyl group, or an active ester group. When immobilizing a polypeptide, it is preferable to introduce an amino group, a carbohydride group, an epoxy group, a formyl group, a metal chelate or an active ester group. When a carrier into which a metal chelate has been introduced is used, a polypeptide having a label having an affinity for a metal ion such as a polyhistidine sequence can be immobilized effectively and stably.

The method for immobilizing the bio-related molecule on the carrier of the present invention is not particularly limited. For example, a biologically relevant molecule can be immobilized on the surface of the carrier by dissolving the biologically relevant molecule in a buffer to prepare a solution and immersing the carrier as described above. Immersion is usually performed at 0 to 98 ° C, preferably 4 ° C to 50 ° C, usually 1 minute to 24 hours, preferably 10 minutes to 1 hour. In this case, the biologically relevant molecules that are not immobilized can be removed by immersing for a certain period of time and then washing the carrier. Also spotter By using a single device, many types of biological molecules can be immobilized on the surface of the carrier. In the case of using a spotter, for example, a biologically relevant molecule solution is spotted on a carrier with a spotter, followed by baking in a heated oven for a certain period of time, and then unfixed molecules are removed by washing. By using a spotter device, other types of bio-related molecules can be immobilized at different positions on the carrier, so many tests can be performed at once. Example

 Hereinafter, the present invention will be described in more detail with reference to examples. However, the technical scope of the present invention is not limited to the following examples.

Example 1

 The carrier support member shown in FIGS. 1a and 1b was inserted into a hollow holder containing the reaction solution. The results are shown in Fig. 6 a and b, respectively. In FIG. 6a, the rear end portion of the carrier support member and the edge of the hollow holder opening are engaged to position the carrier support member so as to be inserted into the central portion of the hollow holder. The liquid level of the substrate is horizontal and the carrier is in contact with the reaction solution. In FIG. 6b, since the rear end portion of the carrier support member and the edge of the hollow holder opening are not engaged with each other, the carrier support member is inserted to the right in the hollow holder. As a result, the surface of the reaction solution is tilted by the surface tension, and there is a portion where the carrier and the reaction solution are not in contact.

Example 2

 Using a ionized vapor deposition method on a 3 mm square silicon substrate, two DLC layers were formed under the following conditions.

1st layer • 2nd layer

Source gas CH ₄ 4. 75 47.5, sscm

H ₂ 0. 25 2. 5 ossein)

 Working pressure 3. 0 8. 0 (Pa)

 Substrate bias DC voltage 500 500 (V)

 High frequency output 100 ― (W)

 Anode voltage 50 50 (V)

 Filament voltage 7 7 (V)

¾ί¾Ε 22 22 (A) An amino group was introduced onto a silicon substrate having a DLC layer on the surface using an ammonia plasma under the following conditions. Source gas H ₃ 30 (sscm) Operating pressure 8.0 ^ sscm) Substrate bias DC voltage 500 (Pa)

 High frequency output ― (w)

 Anode voltage 50 (V)

 Filament voltage 7 (V)

 Tortoise 22 (A)

1-methyl- with 14 OmM succinic anhydride and 0.1M sodium borate

It was immersed in a 2-pyrrolidone solution for 30 minutes to introduce carboxyl groups. 0.1M potassium phosphate buffer, 0.1M 1- [3- (dimethylamino) propyl] solution in a solution containing 3-ethyl chloride, 2 OmM N-hydroxysuccinimide

It was immersed for 30 minutes for activation, and a carrier having a DLC layer and an N-hydroxysuccinimide group as a chemical modifying group on the silicon substrate surface was obtained.

The DNA probe was dissolved in Ο μΟ with Sol. 6 (Toyo Kohan Co., Ltd.) and spotted on a carrier. After baking at 80 ° C for 1 hour, washing with 2 XS SCZ0.2% SDS, washing with ultrapure water and centrifugal drying, the DNA probe was immobilized on the carrier. The region hybridizing with the probe was amplified by PCR. Labeling was performed using CyDye. The composition of the PCR solution was as follows. A sample was prepared by dissolving 30 μl of the obtained CRC product in a hybrid solution (4 XS SCZO. 2% SDS solution) 30 1. A sample of 50 μ1 was placed in the hollow holder shown in Fig. 7c. The carriers on which the DNA probes obtained above were immobilized were immobilized on the carrier support members shown in FIGS. 7a and 7b, respectively, and inserted into the hollow holders containing the samples. The carrier support member shown in FIG. 7a has a waste liquid groove, but the carrier support member shown in FIG. 7b does not have a waste liquid groove. After inserting the carrier support member, react for 2 minutes at 55 ° C, 1 time with 2 XS SCZ 0.2% 505, 1 time with 1 N sodium acetate 0.5% twe 20 1, 1 NM g C 1 ₂ / 0. Washed once with 5% twe en 20. Using a cooled C CD camera, the living body interacting on the carrier via a fluorescent filter (for Cy 5; manufactured by Edmond) Fluorescent labels of related molecules were detected. <i> Excitation light was irradiated at an angle of 50 ° with respect to the support surface using a 5 mm laser (640 nm wavelength). The results are shown in FIG.

 When a carrier support member having a waste liquid groove (FIG. 7a) was used, the liquid could not be accumulated in the carrier fixing part and could be detected well (FIG. 8a). On the other hand, when the carrier support member without the waste liquid groove (Fig. 7b) was used, a partial liquid pool was observed (Fig. 8b). Example 3

 In the posture inserted into the hollow holder, the carrier supporting member (Fig. 9a) in which the volume ratio of the carrier supporting member to the volume of the hollow holder is 80% and the carrier supporting member (Fig. 9) is 50%. b) was inserted into each of the hollow holders containing the reaction solution. In the case of Fig. 9a, bubbles are generated at the time of insertion, but in the case of Fig. 9b, bubbles are not generated at the time of insertion, indicating that the contact between the reaction solution and the carrier is good.

 All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety. Explanation of symbols

 1 1: Hollow holder, 1 2: Carrier support member, 1 3: Rear end of carrier support member, 14: Carrier, 15: Hollow holder opening, 2 1: Hollow holder axis, 2 4: Carrier support 3 1: carrier fixing part, 3 2: bottom surface of carrier fixing part, 3 3: side surface of carrier fixing part (inclined surface), 3 4: angle formed between bottom surface and side surface (inclining surface), 4 1 : Waste liquid groove

Claims

The scope of the claims 1. A unit for use in detecting the interaction of biological molecules using a carrier on which biological molecules are immobilized,  A hollow holder having an opening at one end and the other end closed; and a carrier supporting member on which a carrier to which a biological molecule is immobilized is immobilized and can be inserted into the hollow holder. In a posture in which the member is inserted into the hollow holder, the rear end portion of the carrier supporting member and the edge of the opening of the hollow holder are engaged, whereby the hollow holder is sealed and the carrier supporting member and the hollow holder are Positioned,  In the sectional view taken along the plane including the axial center of the hollow holder in the positioning posture, the area defined by the inner side of the hollow holder and the outer side of the carrier supporting member to which the carrier is fixed is the carrier supporting member. In the region from the carrier fixing part to the tip part, it is substantially the same on the left and right sides of the shaft center. 2. The unit according to claim 1, wherein the carrier fixing portion in the carrier supporting member is a concave portion having a bottom surface and a side surface, and the carrier is disposed on the bottom surface of the concave portion. 3. The unit according to claim 2, wherein at least the side surface on the rear end side of the concave portion of the carrier support member is an inclined surface. 4. The unit according to claim 3, wherein the surface roughness of the inclined surface is 10 / i in or less, and the angle formed by the inclined surface and the bottom surface is 75 ° or less. 5. The unit according to any one of claims 2 to 4, wherein a waste liquid groove is formed from the side surface on the tip end side of the carrier support member recess to the tip end portion. 6. In the posture in which the carrier support member is inserted into the hollow holder, the ratio of the volume of the carrier support member to the volume of the hollow holder in the region closer to the tip than the carrier fixing portion of the carrier support member is 60% or more. The unit according to any one of claims 1 to 5, wherein: 7. In the posture in which the carrier support member is inserted into the hollow holder, the volume ratio of the carrier support member to the volume of the hollow holder is 25 to 70%. Any of the items listed in item 1. 8. Each of the carrier support members has a plurality of carrier support members fixed thereto, each of the rear end portions of the carrier support member is fixed to a face material, and each of the support members has a plurality of hollow holders. The unit according to any one of claims 1 to 7. 9. A method for detecting an interaction of a biological molecule using a carrier on which the biological molecule is immobilized, comprising:  By inserting a carrier support member on which a carrier on which a biologically relevant molecule is immobilized is inserted into a hollow holder having an opening at one end and the other end closed and containing a reaction solution, An interaction step in which the biologically relevant molecule on the carrier interacts with the fluorescently labeled biologically relevant molecule in the reaction solution,  A washing step for removing the biologically relevant molecule that did not interact with the biologically relevant molecule immobilized on the carrier by washing the carrier;  Detection process that irradiates the carrier with excitation light and detects fluorescence with a detector Including  In a posture where the carrier support member is inserted into the hollow holder, the rear end portion of the carrier support member and the edge of the opening of the hollow holder are engaged, whereby the hollow holder is sealed and the carrier support member and the hollow holder are Is positioned,  In the sectional view taken along the plane including the axis of the hollow holder in the positioning posture, the area defined by the inner side of the hollow holder and the outer side of the carrier supporting member to which the carrier is fixed is the carrier of the carrier supporting member. In the region from the fixed part to the tip part, the method is substantially the same on the left and right sides of the axis. 10. The method according to claim 9, wherein the carrier fixing portion in the carrier supporting member is a concave portion having a bottom surface and a side surface, and the carrier is disposed on the bottom surface of the concave portion. 1 1. The method according to claim 10, wherein at least a side surface of the concave portion of the carrier support member on the rear end side is an inclined surface. 1 2. The method according to claim 11, wherein the surface roughness of the inclined surface is 10 μm or less, and the angle between the inclined surface and the bottom surface is 75 ° or less. 1 3. The method according to any one of claims 10 to 12, wherein a waste liquid groove is formed from the side surface on the front end side of the concave portion of the carrier support member to the front end portion. 14. In the posture in which the carrier support member is inserted into the hollow holder, the ratio of the volume of the carrier support member to the volume of the hollow holder is 60% or more in the region closer to the tip than the carrier fixing portion of the carrier support member. The method according to any one of claims 9 to 13. 1 5. Any one of claims 9 to 14, wherein the carrier support member is inserted into the hollow holder, and the volume ratio of the carrier support member to the volume of the hollow holder is 25 to 70%. The method according to 1. 1 6. Each has a plurality of carrier support members to which the carrier is fixed, each rear end of the carrier support member is fixed to a face material, and each has a plurality of hollow holders corresponding to each support member. The method according to any one of claims 9 to 15.